6-Pyruvoyltetrahydropterin Synthase Deficiency: Review and Report of 28 Arab Subjects

Home , Hyperphenylalaninemia, Sepiapterin reductase

Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch

Year: 2019

Almannai, Mohammed ; Felemban, Rana ; Saleh, Mohammed A ; Faqeih, Eissa A ; Alasmari, Ali ; AlHashem, Amal ; Mohamed, Sarar ; Sunbul, Rawda ; Al-Murshedi, Fathiya ; AlThihli, Khalid ; Eyaid, Wafaa ; Ali, Rehab ; Ben-Omran, Tawfeg ; Blau, Nenad ; El-Hattab, Ayman W ; Alfadhel, Majid

Abstract: BACKGROUND: Tetrahydrobiopterin is an essential cofactor for the hydroxylation of aromatic amino acids phenylalanine, tyrosine, and tryptophan. Therefore, tetrahydrobiopterin deficiency results in hyperphenylalaninemia as well as dopamine and serotonin depletion in the central nervous system. The enzyme 6-pyruvoyltetrahydropterin synthase catalyzes the second step of de novo synthesis of tetrahydrobiopterin, and its deficiency is the most frequent cause of tetrahydrobiopterin metabolism disorders. METHOD: We conducted a retrospective chart review of 28 subjects from 24 families with molecularly confirmed 6-pyruvoyltetrahydropterin synthase deficiency from six centers in three Arab countries. We reviewed clinical, biochemical, and molecular data. We also reviewed previously published cohorts of subjects with 6-pyruvoyltetrahydropterin synthase deficiency. RESULTS: Similar to previous observations, we show that early treatment (less than two months) is associated with better outcome. We identify eight PTS variants in 24 independent families. The most common variant is (c.238A>G; p.M80V) with an allele count of 33%. We also identify one novel variant (c.2T>G; p.?). CONCLUSION: The deficiency of 6-pyruvoyltetrahydropterin synthase is relatively common in the Arab population and should be considered in individuals with hyperphenylalaninemia. More natural history studies with comprehensive biochemical and molecular genetics data are needed for a robust base for the development of future therapy.

DOI: https://doi.org/10.1016/j.pediatrneurol.2019.02.008

Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-180681 Journal Article Published Version

The following work is licensed under a Creative Commons: Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) License.

Originally published at: Almannai, Mohammed; Felemban, Rana; Saleh, Mohammed A; Faqeih, Eissa A; Alasmari, Ali; Al- Hashem, Amal; Mohamed, Sarar; Sunbul, Rawda; Al-Murshedi, Fathiya; AlThihli, Khalid; Eyaid, Wafaa; Ali, Rehab; Ben-Omran, Tawfeg; Blau, Nenad; El-Hattab, Ayman W; Alfadhel, Majid (2019). 6-Pyruvoyltetrahydropterin Synthase Deficiency: Review and Report of 28 Arab Subjects. Pediatric Neurology, 96:40-47. DOI: https://doi.org/10.1016/j.pediatrneurol.2019.02.008

2 Pediatric Neurology 96 (2019) 40e47

Contents lists available at ScienceDirect

Pediatric Neurology

journal homepage: www.elsevier.com/locate/pnu

Original Article 6-Pyruvoyltetrahydropterin Synthase Deﬁciency: Review and Report of 28 Arab Subjects

Mohammed Almannai, MD a, Rana Felemban, MD a, Mohammed A. Saleh, MD a, Eissa A. Faqeih, MD a, Ali Alasmari, MD a, Amal AlHashem, MD b, c, Sarar Mohamed, MD b, Rawda Sunbul, MD d, Fathiya Al-Murshedi, MD e, Khalid AlThihli, MD e, Wafaa Eyaid, MD f, Rehab Ali, MD g, Tawfeg Ben-Omran, MD g, Nenad Blau, MD, PhD h, i, * Ayman W. El-Hattab, MD j, k, Majid Alfadhel, MD f, l, m, a Section of Medical Genetics, Children's Hospital, King Fahad Medical City, Riyadh, Saudi Arabia b Department of Pediatric, Prince Sultan Medical Military City, Riyadh, Saudi Arabia c Department of Anatomy and Cell Biology, College of Medicine, Alfaisal University, Riyadh, Saudi Arabia d Pediatrics Medical Genetic Unit (PMGU), Pediatrics Department, Qatif Central Hospital, Qatif, Saudi Arabia e Department of Genetics, College of Medicine, Sultan Qaboos University, Muscat, Sultanate of Oman f Division of Genetics, Department of Pediatrics, King Abdulaziz Medical City, Ministry of National Guard-Health Affairs (MNGHA), Riyadh, Saudi Arabia g Clinical and Metabolic Genetics Section, Department of Pediatrics, Hamad Medical Corporation, Doha, Qatar h Dietmar-Hopp-Metabolic Center, University Children's Hospital, Heidelberg, Germany i Division of Metabolism, University Children's Hospital Zurich, Switzerland j Department of Clinical Sciences, College of Medicine, University of Sharjah, Sharjah, United Arab Emirates k Genetics Clinics, KidsHeart Medical Center, Dubai, United Arab Emirates l King Abdullah International Medical Research Center (KAIMRC), Riyadh, Saudi Arabia m College of Medicine, King Saud Bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia article info abstract

Article history: Background: Tetrahydrobiopterin is an essential cofactor for the hydroxylation of aromatic amino acids Received 18 September 2018 phenylalanine, tyrosine, and tryptophan. Therefore, tetrahydrobiopterin deficiency results in hyper- Accepted 10 February 2019 phenylalaninemia as well as dopamine and serotonin depletion in the central nervous system. The enzyme Available online 18 February 2019 6-pyruvoyltetrahydropterin synthase catalyzes the second step of de novo synthesis of tetrahydrobiopterin, and its deficiency is the most frequent cause of tetrahydrobiopterin metabolism disorders. Keywords: Method: We conducted a retrospective chart review of 28 subjects from 24 families with molecularly Phenylketonuria confirmed 6-pyruvoyltetrahydropterin synthase deficiency from six centers in three Arab countries. We Tetrahydrobiopterin PTPS deficiency reviewed clinical, biochemical, and molecular data. We also reviewed previously published cohorts of fi Inborn errors of metabolism subjects with 6-pyruvoyltetrahydropterin synthase de ciency. Results: Similar to previous observations, we show that early treatment (less than two months) is associated with better outcome. We identify eight PTS variants in 24 independent families. The most common variant is (c.238A>G; p.M80V) with an allele count of 33%. We also identify one novel variant (c.2T>G; p.?). Conclusion: The deficiency of 6-pyruvoyltetrahydropterin synthase is relatively common in the Arab population and should be considered in individuals with hyperphenylalaninemia. More natural history studies with comprehensive biochemical and molecular genetics data are needed for a robust base for the development of future therapy. © 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Sciences; Division of Genetics; Department of Pediatrics; King Abdulaziz Medical Funding: No funding was associated with this study. City; Ministry of National Guard-Health Affairs (MNGHA); Riyadh, Saudi Arabia. Competing interests: The authors declare that they have no competing interests. E-mail address: [email protected] (M. Alfadhel). * Communications should be addressed to: Alfadhel; King Abdullah International Medical Research Center (KAIMRC); King Saud Bin Abdulaziz University for Health https://doi.org/10.1016/j.pediatrneurol.2019.02.008 0887-8994/© 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). M. Almannai et al. / Pediatric Neurology 96 (2019) 40e47 41

Introduction In addition to its role in phenylalanine hydroxylation, BH4 is also an essential cofactor for tyrosine and tryptophan hydroxylases, Phenylketonuria (PKU; OMIM #261600), which was first re- which are rate-limiting enzymes in catecholamine and serotonin ported by Asbjorn€ Folling€ in 1934, is one of the most common biosyntheses, respectively. Therefore, besides HPA, BH4 deficiency inborn errors of metabolism with an estimated prevalence of 1 in also results in dopamine and serotonin depletion in the central 10,000 in the European population1. PKU was the first disorder for nervous system.8 This accounts for the progressive neurological which newborn screening was established through bacterial inhi- deterioration in that subset of individuals with HPA despite early bition assay by Robert Guthrie and Ada Susi in 1963.2 PKU is caused dietary management. Finally, BH4 is also an essential cofactor for by the deficiency of phenylalanine hydroxylase, which catalyzes the the three isoforms of nitric oxide synthase. hydroxylation of phenylalanine to generate tyrosine (Fig). As a Disorders of BH4 metabolism account for only 1% to 2% of pa- result, phenylalanine will accumulate to toxic levels causing irre- tients with HPA in Europeans,8 whereas they are more common in versible intellectual disability (ID). some other ethnic groups. For example, BH4 deficiency accounts for Hydroxylation of phenylalanine to tyrosine through phenylala- more than 10% of HPA patients in some countries in East Asia9,10 nine hydroxylase requires the essential cofactor tetrahy- reaching up to one-third of cases in some reports.11 In a cross- drobiopterin (BH4) that was first identified in the 1960s.3 More sectional study from Iran, 76 of 617 (12%) with HPA have BH4 de- than a decade later, a subgroup of individuals with PKU who ficiencies.12 In one old report from south Brazil, PTPS deficiency developed progressive neurological deterioration despite early di- alone represents 17% of cases with HPA.13 etary management was identified.4 The term malignant hyperphenylalaninemia was then used to describe subjects who were Methods found to have deficiency in BH4, the cofactor for phenylalanine hydroxylase.5 We reviewed the electronic and paper medical records of 28 BH4 is synthesized de novo from guanosine-50-triphosphate (GTP) subjects with molecularly confirmed PTPS deficiency from four through a sequence of three reactions carried out by guanosine-50- centers in Saudi Arabia, one center in Oman, and one center in triphosphate cyclohydrolase I, 6-pyruvoyltetrahydropterin synthase Qatar. We used a case report form to collect the demographic (PTPS), and sepiapterin reductase. During hydroxylation of aromatic characteristics of the subjects, the age and pattern of the initial amino acids, BH4 is oxidized to pterin-4a-carbinolamine. BH4 is then presentation, the clinical phenotype, and the biochemical, radio- recycled through the action of two enzymes, pterin-4a-carbinolamine logical, and molecular features. Literature review was conducted dehydratase and dihydropteridine reductase6 (Fig). Defects in any of using PubMeb search (https://www.ncbi.nlm.nih.gov/pubmed/). these enzymes will result in BH4 deficiency. Sepiapterin reductase The nomenclature of variants is according to the Human Genome deficiency and autosomal dominant form of guanosine-50-triphos- Variation Society recommendations. This study was approved by phate cyclohydrolase I deficiency present, however, without hyper- King Fahad Medical City Institutional Review Board (Institutional phenylalaninemia (HPA).7 Review Board registration number H-01-R012).

FIGURE. Biosynthesis of BH4 and consequences of defect in PTPS. AADC, aromatic L-amino acid decarboxylase; DHPR, dihydropteridine reductase; GTPCH, GTP cyclohydrolase I; 5- HIAA, 5-hydroxyindoleacetic acid; HVA, homovanillic acid; NOS, nitric oxide (NO) synthase; PCD, pterin-4a-carbinolamine dehydratase; PAH, phenylalanine hydroxylase; PTPS, 6- pyruvoyltetrahydropterin synthase; SR, sepiapterin reductase; TH, tyrosine hydroxylase; TPH, tryptophan hydroxylase. 42 M. Almannai et al. / Pediatric Neurology 96 (2019) 40e47

Results and discussion TABLE 1. Patient Cohort Characteristics

Local experience: report of 28 Arab subjects Variable Value

Number of subjects 28 A total of 28 individuals with PTPS deficiency have been iden- Male: female (%) 46:54 tified and included in this study. All are Arabs, but they belong to Consanguinity 25 /28 (89%) Ascertainment Family history: 1 (3%) different geographical regions including Saudi Arabia (n ¼ 21), * Oman (n ¼ 5), Egypt (n ¼ 1), and Sudan (n ¼ 1). There is an equal Positive newborn screening: 10 (36%) Symptomatic: 17 (61%) distribution of males and females (46%:54%). Most individuals in Age at presentation in subjects Neonatal period-9 mo our cohort were born to consanguineous parents (89%). Prematu- diagnosed symptomatically (mean 2.6 mo, median 1.5 mo) rity was evident in 15% of the subjects, whereas 40% were born (n ¼ 17) small for gestational age. The birth weight range was 1.25 to 3.0 kg Presenting complaints Hypotonia (11/20, 55%), seizures (5/20; 25%), lethargy and decreased activity (mean 2.3, median 2.25). Microcephaly was evident in 10 of 20 (4/20; 20%), irritability (4/20; 20%), subjects (50 %). oculogyric crisis (3/20; 15%), Ten subjects were diagnosed by newborn screening (NBS), 17 developmental delay (3/20; 15%) were symptomatic, and the remaining one was diagnosed early Initial phenylalanine level 201-2665 (mmol/L) (n ¼ 24) (mean 1120, median 1037) because of family history. Some of those who were diagnosed by Age at start of treatment Diagnosed by NBS: 2 d-2 mo NBS were already symptomatic in the neonatal period (mean 27 d, median 22 d) (Supplementary Table 1). The age range of diagnosis for those Diagnosed symptomatically: 1 mo-3 yr diagnosed symptomatically was neonatal period to 9 months (mean 11.5 mo, median 10 mo) (mean 2.6 months, median 1.5 months). Among subjects who Age at last follow-up 5 mo-18 yr (mean 5.1 yr, median 4.4 yr) presented with early symptoms, common presenting complaints Most recent phenylalanine 26-1677 include hypotonia (11 of 20: 55%), seizures (five of 20: 25%), and level (mmol/L) (mean 156, median 62) lethargy and irritability (four of 20: 20%). The age range of initiation Most recent prolactin 368-2890 of treatment for those diagnosed with NBS was two days to two level (mIU/L) (mean 876, median 736) months (mean 27 days, median 22 days), and for the rest of sub- Treatment BH4 (mg/kg/day) (n ¼ 27) 3.2-2.0 (mean 11 and median 10) jects, it was one month to three years (mean 11.5 months, median L-Dopa (mg/kg/day (n ¼ 27) 2.0 -15.0 (mean 7.7, median 6.3) 10 months). 5-OH-Trp (mg/kg/day) (n ¼ 19) 1.6 -10.0 (mean 5.5, median 6) In regard to the biochemical profile on presentation, phenylal- Last clinical evaluation anine levels ranged between 201 and 2665 mmol/L (mean 1111, Global developmental delay or ID 18/27 (67%) Mid developmental delay 5/27 (18%) median 1039) in subjects diagnosed by NBS, and it was comparable Normal development 4/27 (15%) to those of subjects diagnosed symptomatically, 360 to 2373 mmol/L Truncal hypotonia 14/28 (50%) (mean 1128, median 1037 [P ¼ 0.95]). Pterin analysis on presenta- Seizures 13/28 (46%) tion is available for eight subjects (seven urine samples and one dry Movement disorders 9/27 (33%) blood spot). All but one subject (subject #20) had high neopterin Microcephaly 12/24 (50%) * (range 3.9 to 29.3 mmol/mol Crea, mean 13.2, median 13.4; refer- 3 subjects with abnormal NBS had early symptoms before diagnosis was made; ence range 1.1 to 4), whereas biopterin and biopterin/total pterins see Supplementary Table 1. ratio were low in all of them (Supplementary Table 1). In three additional subjects, only the ratio is available, and it was low in all. On the other hand, the outcome is better in subjects who were Only two subjects (subjects #11 and #21) had cerebrospinal fluid treated early ( 2 months) (n ¼ 14). Four of them have normal (CSF) studies available on presentation, and they showed typical development, and five more have mild delays only. The remaining pattern with high neopterin, low biopterin, low 5- five subjects have global developmental delay; two of them have hydroxyindoleacetic acid (5-HIAA), and low homovanillic acid had IQ testing performed, and it was in the mild ID range (59 and (HVA). Plasma prolactin is available for 17 individuals on presen- 62). Peripheral hypertonia is commonly observed in our cohort (15 tation (range 368 to 2890 mIU/L; mean 876, median 736). of 25; 60%), whereas truncal hypotonia is evident in half of the The age range for individuals in this cohort at their last follow- subjects. Seizures are observed in 13 subjects (46%). Nine subjects up visit was five months to 18 years (mean 5.1 years, median (33%) have movement disorders. Failure to thrive is observed in 14 4.4 years). Phenylalanine levels on last follow-up ranged between subjects (52%), whereas 50% of them have microcephaly. Additional 26 and 1677 mmol/L (mean 156, median 62) (note: the subject who clinical characteristics are summarized in Table 1. had a value of 1677 was known to be noncompliant). Follow-up CSF Neuroimaging is available for nine subjects only, and it is normal studies on last follow-up are available for only two subjects (sub- in five of them (obtained once, not repeated). Findings in the other jects #2 and #4, Supplementary Table 1). Plasma prolactin levels on four subjects include abnormal T2 hyperintensities within the last follow-up ranged between 25 and 10,788 (mean 1461, median posterior tegmental structures of the pons (subject #6, obtained at 476). age two months), mild white matter volume loss along bilateral In regard to treatment, all subjects but one (subject #20, non- centrum semiovale (subject #12, obtained at six years), and white compliant) are on BH4 (dose range 3.2 to 21 mg/kg/day, mean 11, matter changes with mild cerebral atrophic changes (subjects #22 median 10) and L-dopa (dose range 2 to 15 mg/kg/day (mean 7.7, and #27). median 6.3), 19 of 27 are taking 5-hydroxytryptophan (5-OH-Trp) Eight pathogenic variants have been identified in this report for (dose range 1.6 to 10 mg/kg/day; mean 5.5, median 6), and seven 24 independent families (Table 2); all were in homozygous status. subjects are on folinic acid. The most common variant is c.238A>G; p.M80V with an allele Most of our subjects (23 of 27; 85%) show variable degrees of count of 33%, followed by the missense variant (c.342C>G; p.I114M) developmental delay or ID. All subjects who were treated late and the in-frame deletion (c.169_171del; p.V57del), both with an (greater than two months) (n ¼ 13; outcome not available in sub- allele count of 17%. These variants were identified in subjects from ject #28) have global developmental delay or ID, only one of them Saudi Arabia only. One novel variant (subject #6) is identified in this had an intelligence quotient (IQ) testing performed, and it was 50. report; (c.2T>G; p.?) M. Almannai et al. / Pediatric Neurology 96 (2019) 40e47 43

TABLE 2. PTS Variants in This Report

DNA Nucleotide Change Protein Amino Acid Change # Homozygous # Heterozygous Total Allele Count Origin (Families) (Families) (Families)

c.2T>G p.? 1 0 2 Saudi c.155A>G p.N52S 1 0 2 Saudi c.169_171del p.V57del 4 0 8 Saudi c.200C>T p.T67M 2 0 4 Egyptian/Sudanese c.238A>G p.M80V 8 0 16 Saudi c.342C>G p.I114M 4 0 8 Saudi c.367C>T p.P123S 3 0 6 Omani c.400G>A p.E34K 1 0 2 Omani

PTPS deficiency is the most frequent BH4 metabolism disorder common in certain ethnicities. For example, in one study, molecular analysis of the PTS gene was performed on 176 subjects from East Of the BH4 metabolism disorders, PTPS deficiency accounts for Asia, including the Han populations in Taiwan, Mainland China, and more than 60% of cases.14 As of February 13, 2017, there are 1118 Malaysia, as well as the populations of Japan, South Korea, Thailand, subjects in the International Database of Tetrahydrobiopterin De- and the Philippines. Five variants (c.155A>G, c.259C>T, c.272A>G, ficiencies (BIODEF), 735 (66%) of whom have PTPS deficiency c.286G>A, and c.84-291A>G) were the most common, with the first (http://www.biopku.org/biodef/BIODEF_Start.asp). two accounting for 15.6% and 37.5%, respectively, of the allele count. PTPS deficiency is a pan-ethnic disorder. Chinese subjects Two variants (c.58T>C and c.243G>A) were dominant in the represent 51% (372 of 735) of subjects listed in the BIODEF database, Philippines and Okinawa, Japan, respectively, indicating founder whereas Caucasians and Arabs represent 12% (86 of 735) and 8% (57 events restricted to these isolated geographic regions.9 From of 735), respectively. On the other hand, PTPS deficiency is un- PNDdb, two variants (c.260C>T and c.407A>T) are commonly re- common in Africans and Hispanics. None of the subjects listed in ported in Caucasian subjects. In our local cohort, the most common the BIODEF database are of Jewish ancestry. variant is c.238A>G with an allele count of 33%. Interestingly, all There are two recognized forms of PTPS deficiency. The mild or individuals with this variant belong to the same tribe in Saudi peripheral form is characterized by normal levels of CSF neuro- Arabia, indicating a founder effect, which is a common phenome- transmitters in the initial stages, and a good response to BH4 non in Saudi Arabia and Arabs in general.20 The missense variant monotherapy.8 The other form, which is the severe, or the typical, c.200C>T is reported in two individuals here, both originating from form presents early and is characterized by developmental delay/ North Africa (Egyptian and Sudanese). This variant is also reported ID, convulsions, and abnormal tone and movements. In one report, in seven subjects in the PNDdb with different ethnic backgrounds, of 355 subjects with PTPS deficiency, one-fifth presented with the including Caucasian, Chinese, and Tamil. One novel variant is mild “peripheral” phenotype.8 identified in this report: c.2T>G. Pathogenic variants affecting the PTPS deficiency can be diagnosed by finding HPA as part of initiation codon (c.1A>G and c.3G>A) have been reported.9,21 standard newborn screening that is followed by proper evaluation to look for BH4 deficiencies as a potential cause for HPA. Alterna- tively, PTPS deficiency can be diagnosed either in symptomatic Treatment and outcome infants presenting with neurological deterioration who were diagnosed with HPA by NBS but were presumed to have phenyl- There are two main goals of treatment in subjects with PTPS alanine hydroxylase deficiency and treated with diet only or in deficiency: first is controlling HPA with oral BH4 and second is infants who did not have NBS or in whom the screen was false- restoring neurotransmitter homeostasis by administering biogenic negative. ID, hypotonia, and convulsions are the most common amine precursors (L-dopa/carbidopa and 5-OH-Trp). The recom- 8 presenting symptoms in the severe phenotype of PTPS deficiency mended dose range for BH4 is 5 to 10 mg/kg/day, whereas for L- (Table 3). Among disorders of BH4 metabolism, PTPS deficiency has dopa and 5-OH-Trp, the recommended doses are slightly variable the highest risk of prematurity and low birth weights.8 Subjects based on age. For children, the recommend dose is 8 to 15 mg/kg/ with PTPS deficiency have significantly higher phenylalanine con- day and 6 to 9 mg/kg/day, respectively.8 Replacement therapy for centrations compared with other disorders of BH4 metabolism.8 dopamine is more difficult than replacing serotonin, mainly because of the short half-life of L-dopa and the associated adverse advents. In one study, it was shown that administering dopamine Mutation spectrum in the PTS gene and geographical distribution agonist, pramipexole, was associated with a reduction in L-dopa 22 dosage and in potentiating the effects of L-dopa therapy ; high In 1986, 6-pyruvoyltetrahydropterin synthase was purified from doses were associated with impulse control disorders, whereas low 15 23 human liver. Thony et al. cloned and expressed the human cDNAs doses were safe and clinically effective. Long-term L-dopa therapy for the PTPS enzyme, which was subsequently mapped to the may result in low 5-methyltetrahydrofolate levels in CSF, and chromosomal region 11q22.3-q23.3.16 PTS gene spans about eight therefore CSF folate should also be monitored and replaced in kilobases and contains six exons.17 In 1994, Thony et al. character- deficient subjects through folinic acid administration.24 ized, for the first time, three variants in two subjects with PTPS Besides plasma phenylalanine levels and clinical evaluation, deficiency.18 Since then, several pathogenic variants were identi- monitoring the efficacy of therapy in subjects with PTPS deficiency fied, and 141 variants are currently listed in the Database of Gene requires regular measurements of neurotransmitter metabolites (5- Variants Causing BH4 Deficiencies and other pediatric neuro- HIAA and HVA) in CSF. Low CSF5-HIAA and HVA values could be an transmitter disease (PNDdb), distributed across all six exons and indicator for the ongoing developmental impairment.24 In a report five introns (http://www.biopku.org/pnddb/home.asp; accessed on of 36 patients, Jaggi et al. did not find clear correlation between CSF December 10, 2018). No hotspots for mutations are found,19 5-HIAA and HVA values and clinical outcome.24 CSF neurotrans- although some of the reported pathogenic variants are more mitter testing is challenging as it requires invasive procedure and TABLE 3. 44 Summary of Previous Cohorts of Subjects With PTPS Deficiency

Dudesek et al., 2001 Liu et al., 2001 Chien et al., 2001 Lee et al., 2006 Wang et al., 2006 Jaggi et al., 2008

Number of subjects 5 5 10 10 31 26 Ethnicity Turkish and German Chinese Taiwanese Chinese Chinese Mixed Prematurity 2/5 0/5 NA NA NA 4/26 Birth weight (kg) 2.18-3.5 2.7-3 2.66 ± 1.97 NA 3.1 ± 0.5 1.4-3.3 (Mean 2.66, median 2.67) (Mean and median 2.85) 3/10 born SGA (Mean 2.6, median 2.8) Age at diagnosis of PTPS 18 d-7 yr 10 mo-14 yr NA (all diagnosed through NBS) 8 mo-20 yr 2 mo-47.5 mo 1 wk-27 yr deﬁciency (Mean 20 mo, median 3 mo) (Mean 43 mo, median 14 mo) (Mean 6 yr, median 3.5 yr) (Mean 8.6 mo, median 5 mo) (Mean 9 mo, median 1 mo) Presentation Developmental delay/intellectual 1/5 3/5 NA NA 22/26 NA disability Tone abnormalities 3/5 4/5 NA NA 19/26 NA Seizures 1/5 3/5 NA NA 10/26 NA Movement disorders 4/5 0/5 NA NA 11/26 NA Initial biochemical proﬁle Phenylalanine level (mmol/L) 80-1398 236-1210 423-2280 234-2340 181-2054 180-2117 (Mean 635, median 541) (Mean 830, median 938) (Mean 1316, median 1367 (Mean 1211, median 1137) (Mean 964, median 960) (Mean 841, median 713)

Neopterin mmol/mol cr (1.1-4) 1.6-32.8 NA NA 5.92-5.92 1.5 -31 2-40.5 40 (2019) 96 Neurology Pediatric / al. et Almannai M. * (Mean 17.6, median18.7) (Mean 21.1, median 18.8) (Mean 7, median 4.5) (Mean 18, median 17) Biopterin mmol/mol cr (0.5-3) 0-0.65 NA NA 0.005-0.5 0.01-0.81 <0.01-0.56 * (Mean 0.2, median 0.1) (Mean 0.18, median 0.16) (Mean 0.2, median 0.14) Phenotype Severe 4/5 Severe 4/5 NA Severe 8/10 NA NA Mild 1/5 Mild 1/5 Mild 2/10 Treatment BH4 (mg/kg/day) 2.1-6.5 NA 1.50 ± 0.44 NA 1.1-3 3-12 (Mean 3.85, median 3.4) (0.75-2.14) (Mean 1.9, median 1.8) (Mean 6.5, median 6) (4/5 taking) L-Dopa (mg/kg/day) 6.9 and 7.1 NA 8.39 ± 3.67 NA 5.8-12.5 4-18 (2/5 taking) (3.77-14.40) (Mean 9.3, median 8.8) (Mean 9, median 9.5): (22/26 taking) 5-OH-Trp (mg/kg/day) 4.2 and 5.6 NA 2.13 ± 1.76 NA 3.6-6 4-10 (2/5 taking) (0.00-5.32) (Mean 4.7, median 4.3) (Mean 6.8, median 7) (23/26 taking) Developmental delay/ID on 3/5 NA 10/10 10/10 7/26 had IQ less than 70 10/26 last follow-up Mean IQ 76 ± 14 (56-98) IQ (<20-80) (57-115, Mean and

(severity variable) median 80) e 47 Liu et al., 2008 Leuzzi et al., 2009 Opladen et al., 2012 Ye et al., 2013 Fernandez-Lainez et al., 2018 Souza et al., 2018

Number of subjects 12 19 335 240 5 4 Ethnicity Taiwanese Italian Mixed Chinese Mexican Brazilian Prematurity 1/12 1/19 Gestational age 28-37 (Mean 35) NA 2/5 NA Gestational age 35-38 (Mean 37, median 38) Birth weight (kg) 2-3.3 1.02 -3.5 <1.5 (3.6%) Mean (S.D.) birth weight was 1.8-3.3 NA (Mean 2.6, median 2.65) (Mean 2.7, median 2.8) 1.5-2(8.9%) 3.1 (0.5) kgz (Mean 2.47, median 2.55) 2-2.5(25.8%) 3/5 born SGA 2.5-3 (33.3%) - 3-3.4(20.4%) >3.5 (8%) Age at diagnosis of PTPS Neonatal period 8 d-32 yr Mean 1.8 yr 0.5-156 1-38 mo (Mean 17 mo, 5-96 mo deﬁciency Mean age of diagnosis (Mean 25 mo, median (Mean 11.5, median 3) median 5 mo) (Mean 27 and median 4.5) 20.0 (6.3) d 1mo) Presentation Developmental NA 8/19 Neonates 30% y NAx 5/5 4/4 delay/intellectual disability Infants 45%y Children 52%y Tone abnormalities NA 7/19 Neonates 40% y NAx 4/5 4/4 Infants 62%y Children 40%y Seizures NA 2/19 Neonates 10%y NAx 5/5 1/4 Infants 50%y Children 40%y Movement disorders NA 3/19 Neonates 10%y NAx 5/5 0/4 Infants 18%y Children 25%y Initial biochemical Proﬁle Phenylalanine level (mmol/L) 527-3426 151-2120 Severe phenotype 829 (121e2251) 242-2724 419-1027 538-2582 (Mean 1963, median 2002) (Mean 1025, median 1051) Mild phenotype 1111 (41e3805) (Mean 1035, median 974) (Mean 751, median 779) (Mean 1552, median 1544) Neopterin mmol/mol cr (1.1-4) NA 3.5-44.1 Severe phenotype 21 (1.6e134) 0.08-70.7 4.69-11.88 NA (Mean 17, median 13) Mild phenotype (Mean 12.6, median 9.4) (Mean 6.9, median 6.5) 17.1 (1.9e78) Biopterin mmol/mol cr (0.5-3) NA <0.01-0.55 Severe phenotype 0.2 (0e7.8) 0-2.69 0-0.07 NA

Mild Phenotype (Mean 0.24, median 0.13) (Mean and median 0.04) 40 (2019) 96 Neurology Pediatric / al. et Almannai M. 0.3 (0e1.6) Phenotype NA Severe 13 84.9 % severe NA NA NA Mild 6 15.1 % mild Treatment BH4 (mg/kg/day) 2-4 2-21 Neonates 5.6 (1.8e15) NA NA NA Infants 5.3 (0.5e16) Children 5.5 (0.4e20) L-Dopa (mg/kg/day) 10-15.5 1-10 Neonates 5.9 (1e12) NA NA NA (Mean 12, median 11.25 Infants 7.2 (0.1e44) Children 8.6 (0.3e49) 5-OH-Trp (mg/kg/day) 1-5.8 1-8 Neo 4.5 (0.6e10) NA NA NA (Mean 3.9, median 4) Infants 5.4 (0.5e42) Children 6.1 (0.5e37) Developmental delay/ID on last IQ 86-111 8/19 NA IQ 36-11 (Mean 79, median 80); 3/5 NA follow-up (severity variable) (Mean 97, median 95) available for 31 subjects

Abbreviations:

Cr ¼ Creatinine e DHPR ¼ Dihydropteridine reductase 47 GTPCH ¼ GTP cyclohydrolase I ID ¼ Intellectual disability IQ ¼ Intelligence quotient NA ¼ Not available or not applicable NBS ¼ Newborn screening SGA ¼ Small for gestational age * nmol/l y Out of subjects with severe phenotype z Calculations included six subjects with DHPR deficiency and four subjects with GTPCH deficiency (total 250) x 73.6 % of patients presented with typical symptoms of BH4 deficiency (including hypotonia, apathy, drowsiness, low response, drooling, and retardation of motor development), 70.4 % with systemic symptoms of HPA (including yellow hair, white skin, characteristic body odor, convulsions, mental retardation, occasional tremors, and microcephaly), and 4.0 % with other symptoms (including eczema and vomiting). 45 46 M. Almannai et al. / Pediatric Neurology 96 (2019) 40e47 specialized laboratory facilities to analyze the samples, which are Acknowledgement not readily available. This was a challenge that we faced with our local subjects. Alternatively, plasma prolactin levels, which We would like to thank the participating patients and families. correlate inversely with hypothalamic dopamine levels, could be used,25 as dopamine originating from the hypothalamic tuber- oinfundibular tract is the major physiological inhibitor for Supplementary data prolactin.26 Ogawa et al. described an individual with more significant correlation of L-dopa dose with serum prolactin levels Supplementary data to this article can be found at https://doi. than CSF HVA levels.27 Similarly, in another study, monitoring org/10.1016/j.pediatrneurol.2019.02.008. serum prolactin was used successfully for optimizing the dosage 28 of L-dopa. However, prolactin levels do not reflect serotonin References homeostasis and could also fluctuate due to external factors such 29 as exercise and stress. Prolactin secretion has marked diurnal 1. Hardelid P, Cortina-Borja M, Munro A, et al. The birth prevalence of PKU in variations, with lowest levels found shortly after awakening. populations of European, South Asian and sub-Saharan African ancestry living Therefore, this time is chosen for the basal evaluation of prolactin in South East England. Ann Hum Genet. 2008;72:65e71. 26 2. Guthrie R, Susi A. A Simple phenylalanine method for detecting phenylketon- secretion. uria in large populations of newborn infants. Pediatrics. 1963;32:338e343. Developmental outcome is worse in those who are treated late 3. Kaufman S. The structure of the phenylalanine-hydroxylation cofactor. Proc (i.e., greater than two months). In a report of 10 subjects from Natl Acad Sci U S A. 1963;50:1085e1093. – ± 4. Danks DM, Bartholome K, Clayton BE, et al. Malignant hyperphenylalaninaemia Taiwan, the average IQ score was 76 14, and it was inversely current status (June 1977). J Inherit Metab Dis. 1978;1:49e53. 11 correlated to the age of starting medication. A similar observa- 5. Danks DM, Cotton RG, Schlesinger P. Diagnosis of malignant hyper- tion was made in another report.30 Wang et al. showed that sub- phenylalaninaemia. Arch Dis Child. 1979;54:329e330. 6. Thony€ B, Auerbach G, Blau N. Tetrahydrobiopterin biosynthesis, regeneration jects who were diagnosed by neonatal screening had much higher and functions. Biochem J. 2000;347:1e16. development quotient or IQ than those who were diagnosed 7. Blau N, BonafeL,Th ony€ B. Tetrahydrobiopterin deficiencies without hyper- symptomatically (88 versus 62, respectively).30 Leuzzi et al. phenylalaninemia: diagnosis and genetics of dopa-responsive dystonia and fi e showed that only two of eight subjects with late (greater than sepiapterin reductase de ciency. Mol Genet Metab. 2001;74:172 185. 8. Opladen T, Hoffmann GF, Blau N. An international survey of patients with two months) treatment had normal mental development, tetrahydrobiopterin deficiencies presenting with hyperphenylalaninaemia. whereas all the others were neurologically impaired.31 In a Chi- J Inherit Metab Dis. 2012;35:963e973. nese study, the median age at which treatment was started was 9. Chiu Y-H, Chang Y-C, Chang Y-H, et al. Mutation spectrum of and founder ef- fi fects affecting the PTS gene in East Asian populations. J Hum Genet. 2012;57: signi cantly (P value 0.02) less in subjects with an IQ above 70 145e152. than in those with an IQ below 70.32 Tanaka et al. showed that 10. Liu TT, Chiang SH, Wu SJ, Hsiao KJ. Tetrahydrobiopterin-deficient hyper- individuals with PTPS deficiency who were started on treatment phenylalaninemia in the Chinese. Clin Chim Acta Int J Clin Chem. 2001;313: 157e169. after age 2.5 years performed poorly on tests of executive func- 11. Chien YH, Chiang SC, Huang A, et al. Treatment and outcome of Taiwanese tioning, and they hypothesized that there is a critical level during patients with 6-pyruvoyltetrahydropterin synthase gene mutations. J Inherit which adequate neurotransmitter levels, especially dopamine, is Metab Dis. 2001;24:815e823. 33 12. Khatami S, Dehnabeh SR, Zeinali S, et al. Four years of diagnostic challenges required for the stable development of executive functioning. with tetrahydrobiopterin deficiencies in Iranian patients. JIMD Rep. 2017;32: Although early treatment is clearly associated with better out- 7e14. comes, one report showed that early-treated subjects with PTPS 13. Jardim LB, Giugliani R, Coelho JC, Dutra-Filho CS, Blau N. Possible high fre- fi quency of tetrahydrobiopterin deficiency in south Brazil. J Inherit Metab Dis. de ciency may not maintain movement rhythm as well as normal 1994;17:223e229. 34 subjects, even with external cues. 14. Blau N. Genetics of phenylketonuria: then and now. Hum Mutat. 2016;37: 508e515. fi Conclusion and future directions 15. Takikawa S, Curtius HC, Redweik U, Ghisla S. Puri cation of 6-pyruvoyl-tetrahydropterin synthase from human liver. Biochem Biophys Res Commun. 1986;134:646e651. PTPS deficiency is relatively common in the Arab population 16. Thony€ B, Heizmann CW, Mattei MG. Chromosomal location of two human and should be considered in individuals with hyper- genes encoding tetrahydrobiopterin-metabolizing enzymes: 6-pyruvoyl-tetrahydropterin synthase maps to 11q22.3-q23.3, and pterin-4 alpha-carbinol- phenylalaninemia. It is apparent from several studies that early amine dehydratase maps to 10q22. Genomics. 1994;19:365e368. treatment is associated with better outcome, so early diagnosis 17. Kluge C, Brecevic L, Heizmann CW, Blau N, Thony€ B. Chromosomal localization, cannot be stressed enough. Although NBS is becoming widely genomic structure and characterization of the human gene and a retro- pseudogene for 6-pyruvoyltetrahydropterin synthase. Eur J Biochem. available in different areas of the world, still there are missed 1996;240:477e484. cases owing to failure to recognize BH4 deficiencies, including 18. Thony€ B, Leimbacher W, Blau N, Harvie A, Heizmann CW. Hyper- PTPS deficiency,asapotentialcauseforHPA.Therefore,adding phenylalaninemia due to defects in tetrahydrobiopterin metabolism: molecu- fi fi lar characterization of mutations in 6-pyruvoyl-tetrahydropterin synthase. Am metabolites speci cforBH4deciencies could help in early J Hum Genet. 1994;54:782e792. diagnosis. In fact, measuring neopterin and biopterin from direct 19. Thony€ B, Blau N. Mutations in the BH4-metabolizing genes GTP cyclohydrolase blood spot for routine diagnosis of BH4 deficiencies proved to be I, 6-pyruvoyl-tetrahydropterin synthase, sepiapterin reductase, carbinolamine- 35 4a-dehydratase, and dihydropteridine reductase. Hum Mutat. 2006;27: useful in one study. With wide availability of next-generation 870e878. sequencing methods and reduced cost, these methods could 20. Al-Owain M, Al-Zaidan H, Al-Hassnan Z. Map of autosomal recessive genetic also be utilized in newborn screening.36 disorders in Saudi Arabia: concepts and future directions. Am J Med Genet A. e In the near future, more therapeutic options could become 2012;158A:2629 2640. 21. Wang R, Shen N, Ye J, et al. Mutation spectrum of hyperphenylalaninemia available for PTPS deficiency. It was shown very early that gene candidate genes and the genotype-phenotype correlation in the Chinese delivery into primary patients' fibroblasts restored BH4 produc- population. Clin Chim Acta Int J Clin Chem. 2018;481:132e138. 37 22. Porta F, Mussa A, Concolino D, Spada M, Ponzone A. Dopamine agonists in 6- tion. Antisense oligonucleotides were used successfully to induce fi e fi pyruvoyl tetrahydropterin synthase de ciency. Neurology. 2009;73:633 637. pseudoexon exclusion in broblasts of patients with splicing vari- 23. Porta F, Ponzone A, Spada M. Long-term safety and effectiveness of prami- ants and restored PTPS enzyme activity and pterin profile.38 Finally, pexole in tetrahydrobiopterin deficiency. Eur J Paediatr Neurol. 2016;20: given the relatively common prevalence of PTPS deficiency in our 839e842. 24. Jaggi€ L, Zurflüh MR, Schuler A, et al. Outcome and long-term follow-up of 36 area, we need a regional collaborative effort to establish a patients with tetrahydrobiopterin deficiency. Mol Genet Metab. 2008;93: specialized laboratory for BH4 deficiencies. 295e305. M. Almannai et al. / Pediatric Neurology 96 (2019) 40e47 47

25. Dudesek A, Roschinger€ W, Muntau AC, et al. Molecular analysis and long-term 32. Ye J, Yang Y, Yu W, et al. Demographics, diagnosis and treatment of 256 pa- follow-up of patients with different forms of 6-pyruvoyl-tetrahydropterin tients with tetrahydrobiopterin deficiency in mainland China: results of a synthase deficiency. Eur J Pediatr. 2001;160:267e276. retrospective, multicentre study. J Inherit Metab Dis. 2013;36:893e901. 26. Spada M, Ferraris S, Ferrero GB, et al. Monitoring treatment in tetrahy- 33. Tanaka Y, Kato M, Muramatsu T, et al. Early initiation of L-dopa therapy enables drobiopterin deficiency by serum prolactin. J Inherit Metab Dis. 1996;19: stable development of executive function in tetrahydrobiopterin (BH4) defi- 231e233. ciency. Dev Med Child Neurol. 2007;49:372e376. 27. Ogawa A, Kanazawa M, Takayanagi M, Kitani Y, Shintaku H, Kohno Y. A case of 34. Kao C-D, Niu D-M, Chen J-T, et al. Subtle brain dysfunction in treated 6- 6-pyruvoyl-tetrahydropterin synthase deficiency demonstrates a more signif- pyruvoyl-tetrahydropterin synthase deficiency: relationship to motor tasks icant correlation of L-Dopa dosage with serum prolactin levels than CSF and neurophysiological tests. Brain Dev. 2004;26:93e98. homovanillic acid levels. Brain Dev. 2008;30:82e85. 35. Opladen T, Abu Seda B, Rassi A, Thony€ B, Hoffmann GF, Blau N. Diagnosis of tet- 28. Vatanavicharn N, Kuptanon C, Liammongkolkul S, et al. Novel mutation rahydrobiopterin deficiency using filter paper blood spots: further development affecting the pterin-binding site of PTS gene and review of PTS mutations in of the method and 5 years experience. J Inherit Metab Dis. 2011;34:819e826. Thai patients with 6-pyruvoyltetrahydropterin synthase deficiency. J Inherit 36. Chaiyasap P, Ittiwut C, Srichomthong C, Sangsin A, Suphapeetiporn K, Metab Dis. 2009;32(Suppl 1):S279eS282. Shotelersuk V. Massive parallel sequencing as a new diagnostic approach for 29. Lennartsson A-K, Jonsdottir IH. Prolactin in response to acute psychosocial phenylketonuria and tetrahydrobiopterin-deficiency in Thailand. BMC Med stress in healthy men and women. Psychoneuroendocrinology. 2011;36: Genet. 2017;18:102. 1530e1539. 37. Thony€ B, Leimbacher W, Stuhlmann H, Heizmann CW, Blau N. Retrovirus- 30. Wang L, Yu W-M, He C, et al. Long-term outcome and neuroradiological mediated gene transfer of 6-pyruvoyl-tetrahydropterin synthase corrects tet- findings of 31 patients with 6-pyruvoyltetrahydropterin synthase deficiency. rahydrobiopterin deficiency in fibroblasts from hyperphenylalaninemic pa- J Inherit Metab Dis. 2006;29:127e134. tients. Hum Gene Ther. 1996;7:1587e1593. 31. Leuzzi V, Carducci CA, Carducci CL, et al. Phenotypic variability, neurological 38. Brasil S, Viecelli HM, Meili D, et al. Pseudoexon exclusion by antisense therapy outcome and genetics background of 6-pyruvoyl-tetrahydropterin synthase in 6-pyruvoyl-tetrahydropterin synthase deficiency. Hum Mutat. 2011;32: deficiency. Clin Genet. 2010;77:249e257. 1019e1027.